Project 4 contributes to the goals of the Program by: 1. Elucidating the molecular mechanisms underlying checkpoint activation in cells exposed to heat shock, 2. Evaluating acute acidification as a strategy to abrogate heat-induced checkpoint response, and 3. Identifying molecular predictors of response to be incorporated in treatment design. Thus, the results will assist interpretation of findings in Project 1 and will be closely integrated with the goals and findings of Project 3. The project tests hypotheses formulated on the basis of discoveries made during the current funding cycle. These include the observation that heat-induced inhibition of DNA replication has a trans-acting component equivalent to checkpoint activation operating through regulation of the activity of replication protein A (RPA) by nucleolin, a key component of the nucleolus. Nucleolin translocates from the nucleolus into the nucleoplasm, which facilitates interaction with RPA. The observation that inhibitors of casein kinase II cause a similar intranuclear translocation of nucleolin allows the development of mechanistic models that are tested in the following Specific Aims. Specific Aim 1 determines whether nucleolin translocation in heated cells is mediated by alteration of phosphorylation at specific sites caused by CKII inhibition. The hypothesis is that nucleolin is a key regulator of a checkpoint response activated in cells exposed to heat, and that the RPA-nucleolin interaction is facilitated by post-translational modifications of nucleolin. Specific Aim 2 tests the hypothesis that heat-induced inhibition of CKII is caused by dissociation of HSP90 from the CKII complex, if Specific Aim 1 confirms that CKII inhibition underlies the nucleolin response in heated cells. Specific Aim 3 tests the hypothesis that acute acidification compromises nucleolin-based checkpoint response in cells grown either at Ph 7.4, or Ph 6.7. Specific Aim 4 evaluates nucleolin translocation as a determinant of heat response. The hypothesis is that nucleolin translocation is a predictor of cellular response to heat and molecular marker for treatment design. The results of the above experiments will advance our understanding regarding checkpoint activation in heated cells, characterize the role of nucleolin in this response, and evaluate the potential of acute acidification as strategy for sensitizing cells to heat. This information may allow the identification and characterization of sensors of heat damage, and could be applied to the design of novel strategies to treat human tumors by combining radiation and hyperthermia.